Mutations in the parkin gene are the main genetic cause of autosomal recessive Parkinson's disease (PD) and mutations in parkin also play a major role in familial PD. Preliminary studies indicate a potential pivotal role for parkin in the ubiquitin proteasomal pathway (UPP) by functioning as an ubiquitin E3 ligase. Most disease causing mutations of parkin are thought to be loss of function mutations that ultimately lead to the absence of ubiquitination and the subsequent failure of UPP-mediated degradation of parkin substrates. Thus, the abnormal accumulation of parkin substrates is thought to play a role in the demise of substantia nigra dopaminergic neurons in patients with parkin mutations. A number of putative parkin substrates have been identified, but their importance in the pathogenesis of PD due to parkin mutations is not known. We propose to characterize parkin knockout mice to formally test the hypothesis that the absence of parkin function is the cause of PD due to parkin mutations. Furthermore, biochemical and proteomic characterization of the parkin knockout mice may shed light on the substrates that are important in the pathogenesis of PD due to parkin mutations. Accordingly experiments are proposed to further characterize the role of parldn and it's substrates in the pathogenesis of PD. In Specific Aim #1 we will characterize parkin knockout mice. In Specific Aim #2 we will evaluate the sensitivity of parkin knockouts to environmental toxins. In Specific Aim #3 we will evaluate the interaction of parkin with the alpha-synuclein interacting protein, synphilin-1 and determine whether parkin mediates K48 or K63 ubiquitin linkages. In Specific Aim #4 we will determine whether parkin interacts with alpha-synuclein by evaluating of the effect of crossing parkin knockout mice with A53T mutant alpha-synuclein transgenic mice and further evaluate the interaction of parkin with the alpha-synuclein interacting protein, synphilin-1. In Specific Aim #5 we will identify and characterize parkin interacting proteins and identify compensatory changes in parkin knockout mice. Development and characterization of parkin knockout, understanding the relationship of parkin, alpha-synuclein and synphilin- 1 in the pathogenesis of PD may provide insight into the molecular mechanisms by which these gene products induce neuronal damage and may provide novel therapeutics and targets to prevent the toxic effects of these familial associated genes in the degenerative process of PD.